(R)-PFI-2 Is a Potent and Selective Inhibitor of SETD7 Methyltransferase Activity in Cells

(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells

Dalia Barsyte-Lovejoya,1,2, Fengling Lia,1, Menno J. Oudhoffb,1, John H. Tatlockc,1, Aiping Donga, Hong Zenga, Hong Wua, Spencer A. Freemand, Matthieu Schapiraa,e, Guillermo A. Senisterraa, Ekaterina Kuznetsovaa, Richard Marcellusf, Abdellah Allali-Hassania, Steven Kennedya, Jean-Philippe Lambertg, Amber L. Couzensg, Ahmed Amanf, Anne-Claude Gingrasg,h, Rima Al-Aware,f, Paul V. Fishi,3, Brian S. Gerstenbergerj, Lee Robertsk, Caroline L. Bennl, Rachel L. Grimleyl, Mitchell J. S. Braamb, Fabio M. V. Rossib,m, Marius Sudoln, Peter J. Browna, Mark E. Bunnagek, Dafydd R. Owenk, Colby Zaphb,o, Masoud Vedadia,e,2, and Cheryl H. Arrowsmitha,p,2

aStructural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7; bBiomedical Research Centre, University of British Columbia, Vancouver, BC, Canada V6T1Z3; cWorldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, San Diego, CA 92121; dDepartment of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada V6T1Z3; eDepartment of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada M5S 1A8; fDrug Discovery Program, Ontario Institute for Cancer Research, Toronto, ON, Canada M5G 0A3; gCentre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada M5G 1X5; hDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; iWorldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Sandwich, Kent CT13 9NJ, United Kingdom; jWorldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, CT 06340; kWorldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Cambridge, MA 02139; lNeusentis Research Unit, Pfizer Worldwide Research and Development, Cambridge CB21 6GS, United Kingdom; mDepartment of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V6T1Z3; nWeis Center for Research, Geisinger Clinic, Danville, PA 17821; oDepartment of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T1Z3; and pPrincess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, ON, Canada M5G 1L7

Edited by Robert J. Fletterick, University of California, San Francisco School of Medicine, San Francisco, CA, and approved July 22, 2014 (received for review April 22, 2014) SET domain containing (lysine methyltransferase) 7 (SETD7) is impli- do not prematurely develop cancer (17, 18). Thus, the exact func- cated in multiple signaling and disease related pathways with a broad tional role of SETD7 in normal or disease biology is still an open diversity of reported substrates. Here, we report the discovery of app question (10). (R)-PFI-2—a first-in-class, potent (Ki = 0.33 nM), selective, and cell- SETD7 contains a SET [Su(var)3–9 and Enhancer of Zeste- — active inhibitor of the methyltransferase activity of human SETD7 Trithorax] domain that is conserved among many S-adeno- S R and its 500-fold less active enantiomer, ( )-PFI-2. ( )-PFI-2 exhibits an sylmethionine (SAM)-dependent protein lysine methyltransferases unusual cofactor-dependent and substrate-competitive inhibitory (PKMTs) (19). SETD7 has robust monomethyltransferase activity mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adeno- Significance sylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition Protein methyltransferases constitute an emerging but under- for binding to endogenous SETD7 in MCF7 cells pretreated with (R)- characterized class of therapeutic targets with diverse roles in PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocop- normal human biology and disease. Small-molecule “chemical ied the effects of Setd7 deficiency on Hippo pathway signaling, via probes” can be powerful tools for the functional characterization modulation of the transcriptional coactivator Yes-associated protein of such enzymes, and here we report the discovery of (R)-PFI-2—a (YAP) and regulation of YAP target genes. In confluent MCF7 cells, first-in-class, potent, highly selective, and cell-active inhibitor of (R)-PFI-2 rapidly altered YAP localization, suggesting continuous the methyltransferase activity of SETD7 [SET domain containing and dynamic regulation of YAP by the methyltransferase activity (lysine methyltransferase) 7]—and two related compounds for of SETD7. These data establish (R)-PFI-2 and related compounds as control and chemoproteomics studies. We used these com- MEDICAL SCIENCES a valuable tool-kit for the study of the diverse roles of SETD7 in pounds to characterize the role of SETD7 in signaling, in the cells and further validate protein methyltransferases as a drug- Hippo pathway, that controls cell growth and organ size. Our gable target class. work establishes a chemical biology tool kit for the study of the diverse roles of SETD7 in cells and further validates protein epigenetics | chemical biology | chemical probe methyltransferases as a druggable target class.

Author contributions: D.B.-L., M.J.O., J.H.T., M. Schapira, J.-P.L., A.L.C., A.-C.G., R.A.-A., P.V.F., rotein methyltransferases play diverse roles in the epigenetic B.S.G., L.R., C.L.B., R.L.G., P.J.B., M.E.B., D.R.O., C.Z., M.V., and C.H.A. designed research; Pregulation of gene transcription, silencing, chromatin struc- D.B.-L., F.L., M.J.O., A.D., H.Z., H.W., S.A.F., G.A.S., E.K., R.M., A.A.-H., S.K., J.-P.L., A.L.C., ture establishment, maintenance, DNA repair, and replication. SET A.A., and R.L.G. performed research; M.J.S.B., F.M.V.R., and M. Sudol contributed new domain containing (lysine methyltransferase) 7 (SETD7) (SET9; reagents/analytic tools; D.B.-L., F.L., M.J.O., J.H.T., A.D., H.W., M. Schapira, E.K., R.M., A.A.-H., S.K., J.-P.L., A.L.C., A.A., R.A.-A., P.V.F., B.S.G., L.R., C.L.B., R.L.G., D.R.O., C.Z., M.V., and C.H.A. SET7/9, KMT7) was one of the first protein lysine methyltrans- analyzed data; and D.B.-L., M.J.O., J.H.T., M. Schapira, A.-C.G., P.J.B., M.E.B., D.R.O., C.Z., ferases to be discovered and was originally characterized as a mon- M.V., and C.H.A. wrote the paper. omethyltransferase of lysine 4 on histone H3 (H3K4me1) (1, 2). The authors declare no conflict of interest. Subsequently, SETD7 has been shown to have a very broad target This article is a PNAS Direct Submission. specificity in vitro, including transcriptional regulators such as – Data deposition: The atomic coordinates and structure factors have been deposited in the TAF10,p53,ER,p65,STAT3,Rb,Mypt,Tat,andFoxo3(316). Protein Data Bank, www.pdb.org (PDB ID code 4JLG). SETD7 is also reported to regulate DNA methyltransferase 1 1D.B.-L., F.L., M.J.O., and J.H.T. contributed equally to this work. (DNMT1) in a brain-specific manner (14) and functionally interacts 2 β To whom correspondence may be addressed. Email: [email protected], m.vedadi@ with Pdx1 in pancreatic cells (6). The diverse nature of its sub- utoronto.ca, or [email protected]. strates has implicated SETD7 in multiple molecular pathways in- 3Present address: University College London School of Pharmacy, London WC1N 1AX, volved in cancer, metabolism, and inflammation. Despite these United Kingdom. diverse substrates and molecular pathways, mice with a genetic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. deletion of Setd7 have no obvious developmental deficiencies and 1073/pnas.1407358111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1407358111 PNAS | September 2, 2014 | vol. 111 | no. 35 | 12853–12858 Downloaded by guest on September 24, 2021 investigation of the functional biology of these enzymes, as well as the exploration of their potential as therapeutic targets (25). Here, we describe the discovery of (R)-PFI-2, a potent and selective inhibitor of SETD7, and its use in modulation of the Hippo pathway by SETD7 inhibition. Thus, (R)-PFI-2, together with its 500-fold less-active enantiomer (S)-PFI-2 and a biotinylated derivative of (R)-PFI-2, provides a chemical probe tool kit to interrogate the biology of SETD7. Results (R)-PFI-2 Is a Potent, Selective Inhibitor of SETD7 Methyltransferase Activity. The discovery of (R)-PFI-2 (Fig. 1A) was initiated with a high-throughput screen (HTS) of a 150,000-compound subset of Pfizer’s full HTS collection in an in vitro enzymatic assay of recombinant SETD7. After several rounds of structure-guided molecular design, including optimization of cell permeability, the inhibition of SETD7 in vitro catalytic activity was improved >100-fold compared with the initial HTS hit (IC50 of 2.1 μM), resulting in (R)-PFI-2 (synthesis provided in SI Appendix). A robust radioactivity-based assay was used to determine kinetic parameters for methyltransferase activity of SETD7 and to char- Fig. 1. (R)-PFI-2 is a potent inhibitor of SETD7. (A) Chemical structures of acterize inhibitors (Table 1 and SI Appendix,Fig.S1). (R)-PFI-2 SETD7 inhibitors (R)-PFI-2 and its less-active enantiomer (S)-PFI-2. (B) The inhibited the methyltransferase activity of human SETD7 with an effect of (R)-PFI-2 (●) and (S)-PFI-2 (▲) on methyltransferase activity of IC50 value of 2.0 ± 0.2 nM whereas its enantiomer, (S)-PFI-2, was ± ± μ SETD7. Compounds inhibited SETD7 activity with IC50 values of 2.0 0.2 nM 500-fold less active (IC50 value of 1.0 0.1 M), making the latter (Hill slope, 0.8) and 1.0 ± 0.1 μM (Hill slope: 0.7), respectively. All experiments an excellent compound for use as a negative control (24) in were performed in quadruplicate. (C) Effect of (R)-PFI-2 on activity of 18 chemical biology experiments (Table 1 and Fig. 1B). Measurement different protein methyltransferases [(red filled circle) G9a, (blue filled of the fractional velocities as a function of (R)-PFI-2 concentra- square) EZH2, (green filled triangle) EHMT1, SUV39H2, EZH1, SUV420H1, tion yielded a Morrison Kapp (26, 27) value of 0.33 ± 0.04 nM, SUV420H2, SETD8, SETD2, PRMT1, PRMT3, PRMT5, PRMT8, SETDB1, MLL1, i confirming that (R)-PFI-2 potently inhibits SETD7 in vitro (SI DOT1L, WHSC1, and SMYD2] and DNMT1 was assessed using as high as ) > 50 μM(R)-PFI-2. Experiments were performed in triplicate. Appendix,Fig.S1D .(R)-PFI-2 is highly selective ( 1,000-fold) for SETD7, over a panel of 18 other human protein methyltransferases and DNMT1, and was shown to be inactive against on H3K4 N-terminal peptides in vitro, but little or no enzymatic 134 additional ion channel, GPCR, and enzyme targets (<35% activity when nucleosomal H3 is used as the substrate (1, 2). inhibition at 10 μM) (Fig. 1C and SI Appendix, Table S1). Furthermore, knockdown or depletion of SETD7 does not affect the global cellular levels of the H3K4me1 mark (17, 18), raising (R)-PFI-2 Binds in the Substrate-Binding Pocket. To better un- the question of the physiological context of H3 as a substrate (10). derstand the inhibitory mechanism, we solved the X-ray crystal Increasing evidence supports a role for SETD7 in methylation of structure of the human SETD7 catalytic domain bound to diverse nonhistone proteins, especially transcription factors and (R)-PFI-2 at 1.9-Å resolution, revealing that the inhibitor occupies chromatin regulatory complexes, leading to changes in gene- part of the substrate peptide-binding groove of the enzyme expression programs, some of which were shown to involve loci- extending deep into the lysine-binding active site (Fig. 2 and SI specific H3K4 monomethylation (reviewed in ref. 10). Peptide array Appendix, Fig. S2 and Table S2). There are several noteworthy studies in vitro have identified a consensus SETD7 substrate se- features of this interaction. First, an intramolecular pi-stacking interaction between the phenyl group of the inhibitor’s tetrahy- quence found in previously reported SETD7 substrates as well as droisoquinoline core and the trifluoromethylated phenylalanine novel targets (7). Nevertheless, the functional significance of most substructure leads to a compact conformation of the protein- of these targets is largely unexplored. bound inhibitor. Second, (R)-PFI-2 efficiently occupies the In addition to its broad substrate specificity, SETD7 also has portion of the peptide-binding groove that is normally occupied unique features that distinguish it from most other PKMTs. First, by the target lysine residue and the peptide backbone of the SETD7 has neither canonical nuclear localization signals nor preceding two residues of the substrate peptide (Fig. 2 A and C–E). nuclear export signals and has been reported to localize in both These two residues (-1 and -2 relative to substrate Lys) have been the cytoplasm and the nucleus (20, 21). Thus, nuclear localiza- shown to be the most important residues for substrate binding by tion of SETD7 may be regulated, at least in part, via interaction SETD7 (7), suggesting that (R)-PFI-2 will be effective at inhibiting with other cellular factors. For example, SETD7 is recruited to the promoters of target genes by interacting with transcription factors such as NFκB (20). Secondly, SETD7 is the only methyl- Table 1. Enzymatic characterization of wild-type SETD7 and transferase that contains membrane occupation and recognition mutants nexus (MORN) repeats that are found in proteins linking the K app, μM membrane to the cytoskeleton (22). This observation suggests m −1 a potential cytoplasmic role for SETD7. Given the multitude of Protein Peptide SAM kcat,h (R)-PFI-2 IC50, nM* methylation and interaction targets and paucity of distinct SETD7- ± ± ± ± associated phenotypes identified to date, it is likely that SETD7 Wild-type 1.1 0.1 0.3 0.1 48 2 2.0 0.2 ± ± ± ± function may be modulatory, subtle, and highly dependent on the H252W 1.8 0.2 0.5 0.1 37 4 2,630 200 ± ± ± ± cell type and/or physiological conditions being studied. D256A 1.9 0.1 0.4 0.1 7 1 1,200 90 ± ± ± ± Recently, the identification of chemical probes (23, 24)—potent, V274E 3.1 0.2 0.7 0.2 3 0.5 10,770 2,000 selective, cell-active small molecules—for protein methyltransferases *Methods are described in SI Appendix. All experiments were performed in such as G9a, DOT1L, and EZH2 has greatly enabled the triplicate, and values are presented as mean values ± SD.

12854 | www.pnas.org/cgi/doi/10.1073/pnas.1407358111 Barsyte-Lovejoy et al. Downloaded by guest on September 24, 2021 The importance of these residues for inhibitory activity by (R)-PFI-2 was verified by site-directed mutagenesis (Table 1). First, H252 contributes an important hydrogen bond to (R)-PFI-2 binding (Fig. 2B) but does not interact with the H3 peptide substrate (PDB ID code 1O9S) (28). Mutation of H252 to tryptophan resulted in an enzyme that was as active as the wild- type protein for the H3(1–25) substrate, but for which the inhibitory effect of (R)-PFI-2 was reduced more than 1,000-fold (Table 1), confirming that this interaction makes important contributions to the mode of inhibition by (R)-PFI-2. We also evaluated two mutants of residues that are involved in both (R)-PFI-2 and peptide binding, D256A and V274E (Table 1). Although these mutants had no significant change in Km values for SAM and peptide, both had much lower activity (kcat values − of 7 ± 1 and 3 ± 0.5 h 1, respectively). These two mutations also displayed a dramatic increase in IC50 values for (R)-PFI-2, consistent with the mode of binding observed in the crystal structure. These data clearly indicate that (R)-PFI-2 binds within the peptide-binding site with unique interactions that contribute to its high potency, while also interacting with residues that contribute to peptide binding and enzymatic activity. Finally, our structural data also predict that (S)-PFI-2 is unable to form the full set of interactions observed for (R)-PFI-2 (Fig. 2B), explaining its reduced inhibitory activity.

(R)-PFI-2 Is a SAM-Dependent Inhibitor. Our structural data indicate that (R)-PFI-2 (i) occupies the peptide binding groove, (ii)induces conformational alterations in the post-SET loop, and (iii) makes direct contact with SAM, suggesting a peptide-competitive mode of inhibition that may also be dependent on SAM binding. Moreover, it is well-known that SAM binding to SET domain methyltransferases has an important role in folding and stabilizing the post-SET loop (19), and SETD7 has been reported to have Fig. 2. Structure and binding mode of (R)-PFI-2. (A) Molecular graphics of an ordered binding mechanism in which SAM first binds the en- the 1.9-Å crystal structure of (R)-PFI-2 (magenta) bound within the substrate zyme, followed by peptide binding (29). Together these observa- peptide-binding groove of human SETD7 (surface representation in gray). The cofactor, SAM, is in yellow. (B) Detailed interactions between SETD7 tions further hint at a structural interdependence between SAM (green) and (R)-PFI-2 (magenta). Hydrogen bonds are shown as dashed lines. binding and binding of ligands that occupy the peptide-binding (C) Superimposition of (R)-PFI-2 with the SETD7-bound conformation of a groove formed by the post-SET loop. substrate histone peptide (PDB ID code 1O9S) (28), showing partial occu- To investigate in more detail the binding properties of (R)-PFI-2, pation of the peptide-binding site by (R)-PFI-2. (D) Superimposition of we next performed surface plasmon resonance (SPR) experiments, (R)-PFI-2–bound SETD7 (green) with all 23 SETD7 structures in complex with confirming that (R)-PFI-2 binds to SETD7 only in the presence of cofactor (or cofactor mimic, sinefungin) available from the PDB (gray), SAM (Fig. 3A). Fast on and off rates were observed for SAM showing the conformational variability of the post-SET loop. SETD7 residues binding alone (Fig. 3A, green trace) whereas (R)-PFI-2 alone that form H-bonds to (R)-PFI-2 are shown in green and are located in the less

exhibited no binding to SETD7 (Fig. 3A, red trace). Inclusion of MEDICAL SCIENCES conformationally variable region of the protein. (E) Surface representation μ of (R)-PFI-2–bound SETD7 highlighting an induced conformation of the post- 20 M SAM in (R)-PFI-2 binding injections (but no SAM in the SET loop (green) that is not seen in any of the other SETD7 structures. (F) flow/wash buffer) resulted in binding and dissociation curves Molecular surface representation of SAM (yellow) and (R)-PFI-2 (purple), reflecting both SAM and (R)-PFI-2 contributions (Fig. 3A, blue highlighting hydrophobic interactions between the methyl group of SAM trace). In the presence of constant 20 μM SAM in the flow and and the pyrrolidine moiety of the inhibitor. wash buffer, (R)-PFI-2 binds with an on rate of 1.4 ± 0.16 × 106 −1 −1 −1 M ·s and off rate of 0.0058 ± 0.0009 s yielding a KD value of 4.2 ± 0.2 nM (SI Appendix, Fig. S3). Taken together, these data the wide variety of SETD7 substrates. The pyrrolidine amide are consistent with a significant role for SAM in the binding of occupies the lysine-binding channel and makes direct hydrophobic (R)-PFI-2 to SETD7, as observed in our crystal structure, and interactions with the departing methyl group of SAM (Fig. 2 B and suggest that the enzyme-inhibitory kinetics are likely defined by F), further preventing productive interaction of SETD7 with lysine codependence on both cofactor and substrate concentrations. substrates. Third, binding of (R)-PFI-2 induces a unique confor- Finally, to further investigate the mode of action of (R)-PFI-2, mation of the post-SET loop (residues 336–349), which normally we determined the IC values at varying SAM and peptide “ ” 50 forms one wall of the peptide-binding groove of the enzyme. In concentrations (30) (Fig. 3 B and C). A decrease in IC50 values at previous crystal structures of SETD7, the conformation of this fixed peptide concentration (5 μM) and increasing SAM con- post-SET loop is highly variable and often lacks electron density centration indicated an uncompetitive mode of inhibition with (Fig. 2D). However, in the (R)-PFI-2–bound structure, the post- respect to SAM (Fig. 3B), consistent with our SPR data in- SET loop has an optimized shape complementarity and forms dicating that (R)-PFI-2 binds to SETD7 only in the presence of hydrophobic interactions with the trifluoromethyl moiety of SAM (Fig. 3A). At a saturating concentration of SAM (2 μM) (R)-PFI-2 (Fig. 2 D and E). Fourth, a network of hydrogen bonds and increasing concentration of peptide, (R)-PFI-2 displayed an with G336 at the base of the post-SET loop, and with the opposite apparent noncompetitive behavior with respect to peptide. wall of the substrate binding groove (S268, H252, and D256 in the However, the same experiments carried out at SAM concen- structurally invariable I-SET subdomain), anchor the ligand within trations near or below the Km of SAM (0.3 ± 0.1 μM) (Table 1) the peptide-binding site. resulted in a broadly linear increase in IC50 values more consistent

Barsyte-Lovejoy et al. PNAS | September 2, 2014 | vol. 111 | no. 35 | 12855 Downloaded by guest on September 24, 2021 bound to and stabilized SETD7 (SI Appendix, Fig. S4 C and D). To confirm that (R)-PFI-2 binds to endogenous human SETD7 in cells, we used structure-guided molecular design to generate PFI-766 (Fig. 4A), a biotinylated variant of (R)-PFI-2 that retains the ability to bind and inhibit SETD7 (IC50 110 ± 26 nM in our in vitro enzymatic assay). We used PFI-766 (Fig. 4A) immobilized on streptavidin beads to isolate endogenous SETD7 from lysates of MCF7 cells pretreated (for 1 h or 5 h) with increasing doses of (R)-PFI-2 (Fig. 4 B and C). The amount of SETD7 “pulled down” by the beads was largely competed off by both 1-h and 5-h pretreatment of live cells with 5μM(R)-PFI-2. Because the lysed material in these experiments was diluted by ∼15-fold, we esti- mate a cellular IC50 for binding of (R)-PFI-2 to endogenous SETD7 in the submicromolar range (i.e., 15-fold less than in the cell lysate). PFI-766 engagement of endogenous SETD7 was also confirmed by mass spectrometry that supported the high specificity of the compound for endogenous SETD7 (SI Appendix, Table S3). Taken together, these results demonstrate direct binding of endogenous SETD7 by (R)-PFI-2 in a cellular environment.

(R)-PFI-2–Dependent Inhibition of SETD7 Affects Yes-Associated Protein Localization and Phenocopies Setd7 Genetic Deletion. We Fig. 3. (R)-PFI-2 is a SAM-dependent inhibitor of SETD7. (A) Biacore SPR have recently identified a role for SETD7 in Hippo pathway sensorgram of (R)-PFI-2 and SAM binding to SETD7. Three single-cycle ki- signaling in vitro and in vivo (31). The Hippo pathway is an netics runs with five concentrations were performed. Five injections of 20 μM evolutionarily conserved regulator of cellular proliferation, sur- SAM show consistent binding with rapid on and off kinetics (green); 20 μM vival, and organ size (32). Activation of the Hippo pathway by SAM is expected to be saturating as the KD of SAM for SETD7 was de- cell–cell contact results in the phosphorylation and cytoplasmic μ termined to be 1.1 M under these conditions. In the absence of SAM, a five- retention of the transcriptional coactivator Yes-associated pro- point dilution series of (R)-PFI-2 from 7.8 nM to 125 nM exhibited no binding μ tein (YAP), resulting in decreased expression of YAP target to SETD7 (red). When the (R)-PFI-2 injections included 20 M SAM, a visually −/− biphasic binding curve was observed, reflecting fast SAM binding with genes such as Areg, Cdc20, Ctgf, Cyr61, and Gli2.InSetd7 subsequent slower (R)-PFI-2 binding (blue). At the highest (R)-PFI-2 concen- murine embryonic fibroblasts (MEFs), cell–cell contact does not tration (rightmost blue curve), the dissociation curve was single phase lead to YAP sequestration in the cytoplasm, resulting in in- whereas, at lower (R)-PFI-2 concentrations, the dissociation curves were bi- creased levels of nuclear YAP and heightened expression of phasic. Under these latter conditions, SETD7 is essentially saturated with YAP target genes. Although direct methylation of Yap by SAM, but the (R)-PFI-2 concentrations were not fully saturating and the SETD7 was not demonstrated, these effects were dependent on protein is a mix of SAM-bound and SAM plus (R)-PFI-2 bound. This situation the methyltransferase activity of SETD7, as well as a putative gives rise to two visibly different off rates, one for SAM alone and one for the combination of SAM plus (R)-PFI-2. A single-cycle kinetics methodology methylation site (K494) of YAP (31), thus supporting a role for was used. The analysis was done in triplicate using a 1:1 kinetic fitting modelandthedatawereprocessedseparately and values were averaged.

(B)IC50 values were determined for (R)-PFI-2 at varying concentrations of SAM, 5 μM peptide, and 20 nM enzyme. (C)IC50 values at varying concentrations of substrate H3(1–25) peptide and at SAM concentrations of 0.125 (●), 0.25 (■), 0.5 (▲), and 2 μM(▼) using 20 nM enzyme as described in SI Appendix.

with a competitive mode of inhibition with respect to peptide. This effect was most pronounced at the lowest SAM concentrations (Fig. 3C). These data are consistent with our structural observations that (R)-PFI-2 binds to the peptide-binding site but also interacts with the methyl group of SAM. Altogether, our data indicate that inhibition of SETD7 activity by (R)-PFI-2 is unlikely to be purely substrate-competitive in the classical sense; rather, it is compli- cated by the interaction of (R)-PFI-2 with SAM.

(R)-PFI-2 Binds SETD7 in Cells. (R)-PFI-2 has favorable physico- chemical properties, including high aqueous solubility (285 μM), − permeability (Russel–Ralph canine kidney cell = 6.48 × 10 6 cm/sec; LogD7.4 = 2.3), and stability (SI Appendix, Fig. S4A), suggesting that it is an excellent tool for cellular studies of SETD7 catalytic function. (R)-PFI-2 did not affect the viability of four different cell lines treated with (R)-PFI-2 at below 50 μM Fig. 4. (R)-PFI-2 binds to SETD7 in cells. (A) Structure of PFI-766. (B) Sche- (SI Appendix, Fig. S4B). Cellular thermal shift assay (CETSA) matic of the experiment using PFI-766 to pull down endogenous SETD7. (C) PFI-766 pulls down endogenous SETD7 from washed MCF7 cells, and the that measures ligand-induced stabilization of a target protein in ° amount of pulled-down material decreased in a dose-dependent manner in cells showed a 4 C increase in the apparent thermal stability of cells treated with increasing doses of (R)-PFI-2. The control lane of 0 (R)-PFI-2 Flag-tagged SETD7 in HEK293 cells at 10 μM(R)-PFI-2, in- indicates the solvent DMSO control. The gels shown are representative of at dicating that (R)-PFI-2 passed through the cell membrane and least three independent experiments.

12856 | www.pnas.org/cgi/doi/10.1073/pnas.1407358111 Barsyte-Lovejoy et al. Downloaded by guest on September 24, 2021 a novel methylation-dependent checkpoint in activation of the 5 A and B and SI Appendix, Fig. S5C). Furthermore, when we − − Hippo pathway. Consistent with a role for the methyltransferase transfected Setd7 / MEFs with plasmids expressing either + + H252W activity of SETD7 in regulating YAP localization, Setd7 / MEFs SETD7 or SETD7 , we found that nuclear accumulation of grown to confluence in the presence of (R)-PFI-2, but not the YAP after (R)-PFI-2 treatment was inhibited by the H252W less-active enantiomer, displayed increased nuclear localization mutation (SI Appendix, Fig. S5D), consistent with the proposed of YAP as measured by immunofluorescence using two distinct interactions between (R)-PFI-2 and SETD7. Taken together, antibodies (Fig. 5A and SI Appendix, Fig. S5 A and B), as well as these results demonstrate that (R)-PFI-2, through direct interactions with SETD7, modulates YAP localization. increased expression of YAP-dependent genes Ctgf, Gli2, and One of the key advantages of a pharmacological agent over Cdc20 (Fig. 5B and SI Appendix, Fig. S5C). Similar to results in −/− – genetic methods for studying gene/protein function is the ability Setd7 MEFs, (R)-PFI-2 dependent inhibition of SETD7 ac- to study temporal and dynamic processes in cells (24). We tivity had no effect on YAP localization at low cell density when therefore sought to exploit (R)-PFI-2 to interrogate the mecha- Yap is primarily nuclear (Fig. 5A). (R)-PFI-2 had no effect in nistic role of human SETD7 in cells in which the Hippo/YAP −/− Setd7 MEFs, further supporting a SETD7-specific effect (Fig. pathway was already activated. We treated confluent MCF7 cells with (R)-PFI-2 or (S)-PFI-2 for 2 h and examined the subcellular localization of YAP. Strikingly, treatment of high-density MCF7 cells with (R)-PFI-2 resulted in a dose-dependent increase of nuclear YAP (Fig. 5 C–E) and heightened expression of the YAP target genes AREG and CYR61 (SI Appendix, Fig. S5G) whereas (S)-PFI-2 had no effect (SI Appendix, Fig. S5 E and F). These results indicate that YAP localization in confluent MCF7 cells is the net result of a dynamic process that requires constant SETD7 activity. In support of this concept, kinetic analysis showed that inhibition of SETD7 with (R)-PFI-2 led to increased YAP nuclear localization within 30 min of treatment (Fig. 5F), indicating that the effects of SETD7-dependent methylation can be rapidly reversed in cells through inhibition of the enzyme. Together, these results demonstrate SETD7-dependent cellular activity for (R)-PFI-2 and indicate that SETD7-dependent methylation is operating in a continuous and dynamic process to control the subcellular localization and function of YAP fol- lowing activation of the Hippo pathway. Discussion Increasing evidence implicates SETD7 as a regulator of nonhistone proteins, especially transcription factors and chromatin regulators, which in turn modulate gene expression patterns within specific signaling pathways. However, identification of distinct, widely replicated phenotypes associated with these SETD7 activ- ities is still lacking. Studies identifying a role for SETD7 in a wide variety of signaling pathways [e.g., NF-κB (8, 16), p53 (5), STAT3 (15), and ER (13)] have all used knockdown/-out of the entire protein or overexpression in transformed cell lines, which may

obscure or exaggerate subtle regulatory mechanisms. Under ho- MEDICAL SCIENCES − − meostatic conditions, Setd7 / mice fail to support a role in these previously identified pathways (17, 18, 31). However, it is possible that, under specific conditions of stress or inflammation, a more dramatic role for Setd7 in these pathways may emerge. Thus, SETD7-dependent methylation may yet prove to be an important regulatory mechanism and a novel therapeutic target in diseases associated with these important signaling pathways. Here, we report a chemical tool kit for the investigation of Fig. 5. Inhibition of Setd7 affects YAP in MEFs and MCF7 cells. (A) MEFs derived +/+ −/− the molecular and functional role of endogenous SETD7 in from Setd7 or Setd7 mice were grown at low density (LD) or high density cellular signaling and gene expression. (R)-PFI-2 is a potent (HD) in the presence of 10 μM(S)-PFI-2 or (R)-PFI-2. The amount of nuclear YAP ’ was quantified from images as described in SI Appendix.Datafromthree and selective inhibitor of SETD7 s catalytic activity. Its en- experiments were pooled (n > 30; *P < 0.001). (B)(R)-PFI-2 treatment induces antiomer, (S)-PFI-2, is 500-fold less active and serves as YAP target genes in high-density Setd7+/+ MEF cultures. Relative expression is a structurally similar negative control molecule for compari- displayed as the expression levels of Ctgf after (R)-PFI-2 treatment over that with son. PFI-766 is an analog of (R)-PFI-2 that retains strong (S)-PFI-2 treatment prenormalized to the levels of Actb housekeeping gene binding and inhibition and can be derivatized for chemo- (n = 6; *P < 0.05). (C) Confluent MCF7 cultures were treated with (S)-PFI-2 or proteomics and other cellular studies (23). We used these (R)-PFI-2 for 2 h at 1 μM, and stained for the Hippo pathway transducer YAP reagents to demonstrate the specificity of the cellular response, (red) and DAPI (blue). Representative confocal images of equal magnification target engagement in cells, and the pharmacological modulation × μ (150 150 m) are shown. (D) The amount of nuclear YAP quantified from of SETD7 in Hippo signaling in MEFs and MCF7 cells. images in C. Data are from two pooled experiments (n > 30; *P < 0.001). (E) Confluent MCF7 cells were treated with the indicated concentrations of (R)-PFI-2 Our results indicate that SETD7-dependent regulation of for 2 h and show a clear decrease in cytosolic YAP with simultaneous increase YAP localization is a rapid and dynamic process. Of note, it in nuclear YAP with increasing (R)-PFI-2. (F) Confluent MCF7 cultures were in- remains unresolved whether these methylation-dependent cubatedwith1μM(R)-PFI-2 for the indicated times. Western blots of nuclear events are due to direct methylation of the YAP protein itself, or extracts were probed for YAP, SETD7, and ac-H3. whether additional substrates are involved. Thus, given the many

Barsyte-Lovejoy et al. PNAS | September 2, 2014 | vol. 111 | no. 35 | 12857 Downloaded by guest on September 24, 2021 potential pathways in which SETD7 has been implicated in hu- purification, enzymatic mechanism of action studies, biophysical binding man cell signaling (10, 12), (R)-PFI-2 and related compounds measurements, and chemistry methods, are described in SI Appendix. will be valuable tools for the community to better understand Cellular assays of YAP localization were performed in MEFs at both low and SETD7 biology. high density, and confluent MCF7 cells were treated with the indicated concentrations of (R)-PFI-2 or (S)-PFI-2. Nuclear YAP was quantified using ImageJ Finally, our combined structural, biophysical, and enzymatic software using two different YAP antibodies. Further details of image analysis analyses showed that (R)-PFI-2 has a distinctive cofactor-dependent and statistics, along with methods for RNA isolation, Western blots of cytosolic mode of action, combined with a likely substrate-competitive and nuclear protein extracts, and RT-PCR, are described in SI Appendix. contribution. As such, (R)-PFI-2 is the third recent example of highly selective inhibitors that bind in the substrate peptide- ACKNOWLEDGMENTS. We thank members of the M.V. laboratory and the binding groove of SET domain methyltransferases [in addition Pfizer high-throughput screen (HTS) team for assays and HTS. (R)-PFI-2 is to methyltransferase G9a/GLP (33) and SMYD2 inhibitors now commercially available from Tocris. Small amounts of (S)-PFI-2 or PFI-766 are available for research purposes upon request from Structural (34)], suggesting that targeting the peptide-binding groove Genomics Consortium (SGC). The SGC is a registered charity (no. 1097737) may be a general strategy for other human PKMTs, many of that receives funds from AbbVie, Boehringer Ingelheim, the Canada which are attractive therapeutic targets (25, 35). Foundation for Innovation (CFI), the Canadian Institutes of Health Re- search (CIHR), Genome Canada, Ontario Genomics Institute Grant OGI- Materials and Methods 055, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foun- dation, the Ontario Ministry of Economic Development and Innovation, Recombinant human SETD7 (residues 1–366) and SET domain only (residues Pfizer, Takeda, and Wellcome Trust Grant 092809/Z/10/Z. Use of the Ad- 109–366) were expressed in Escherichia coli and purified to homogeneity. vanced Photon Source was supported by US Department of Energy Con- Methyltransferase activity of SETD7(1-366) was assayed using a scintillation tract DE-AC02-06CH11357. This work was supported by CIHR Grants proximity assay monitoring the incorporation of the tritium-labeled methyl MOP-123322 (to A.-C.G.) and MSH-95368, MOP-89773, and MOP-106623 (to C.Z.) and a CFI grant (to C.Z.). C.Z. is a Michael Smith Foundation for group of 3H-SAM into a peptide substrate corresponding to histone H3 – μ μ – Health Research Career Investigator. M.J.O. is the recipient of CIHR/Ca- residues 1 25 at 2 M SAM and 2 M H3(1 25) and 2 nM enzyme (Fig. 1). nadian Association of Gastroenterology/Janssen Inc. and Michael Smith SETD7(109–366) crystallized with (R)-PFI-2 in 23% PEG 3500, 0.1 M lithium Foundation for Health Research postdoctoral fellowships. C.H.A. holds sulfate, and 0.1 M BisTris (pH 6.5). Further details, along with protein a Canada Research Chair in Structural Genomics.

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